The natural pigment carminic acid is one of the most frequently used colorants of food, medicine, cosmetics and textiles.
Carminic acid is a colorant, which can be extracted from the female insect bodies of Dactylopius coccus costa (alternative name Coccus cacti L.). The insects live on Nopalea coccinellifera, Opuntia fidus indica and other plants of the family Cactaceae cultivated for instance in the desert areas of Mexico, Central and South America and Canary Islands. Depending on the pH the colorant may be a color in a spectrum from orange over red to purple and is generally known as cochineal or cochineal color. Carmine colorant is widely used in foods and beverages.
As known in the art Porphyrophora polonica is also producing carminic acid and was cultured for production of carminic acid in e.g. Poland.
In relation to current industrial relevant production, carminic acid is harvested by extraction from the insect's dried bodies with water or alcohol.
The insects (Dactylopius coccus) are cultured on cacti and the supply may therefore be relatively expensive and subject to undesirable variations and price fluctuations.
In order to try to resolve the problem of undesirable variations and price fluctuations—U.S. Pat. No. 5,424,421 (European Colour, published 1995) describes chemical synthesis of carminic acid by a route of synthesis involving different intermediates.
As discussed in e.g. WO02006/056585A1 (Chr. Hansen A/S)—during the aqueous based extraction of carminic acid from the insect, an amount of insect protein is also released from the insect and will be contained in the color extract and it has been reported that the cochineal insect proteins could create some allergy related problems. In WO02006/056585A1 a special process to reduce the amount of insect protein from the insect extract solution is described—however, the final produced color composition/product of WO02006/056585A1 will still comprise some amounts Dactylopius coccus costa insect proteins.
The structure of carminic acid is shown in FIG. 1—as can be seen it is a so-called C-glucoside (i.e. wherein the glucose is joined/conjugated to the aglucon by a carbon-carbon linkage).
According to the art—the term “aglycon” denotes the non-carbohydrate part of the corresponding glycosylated form of the aglycon. When the sugar is glucose the aglycon may be termed aglucon.
According to the art—the term “glycoside” denotes a compound which by hydrolysis results in a sugar and a non-sugar (aglycon) residue, e.g. glucosides can give glucose, galactosides can give galactose. As shown in FIG. 1—hydrolysis of the C-glucoside carminic acid results in glucose and the aglucon kermesic acid (KA).
The in vivo insect (Dactylopius coccus) biosynthetic pathway involved in carmine production is currently not described in details—accordingly, based on the prior art the skilled person does not know which compound is the aglucon during the in vivo Dactylopius coccus biosynthetic production of carminic acid.
Analysis of D. coccus has shown that a broad range of compounds related to carminic acid are present in extract from D. coccus and numerous of these compounds could in principle be glucosylated during the in vivo Dactylopius coccus biosynthetic production of carminic acid.
For instance, the article of Stathopoulou et al (Analytica Chimica Acta 804 (2013) 264-272) describes six new anthraquinones that are present in extract from D. coccus and any of these six new anthraquinones (see e.g. FIG. 1 of the article) could in principle be the molecule which is glucosylated during the in vivo Dactylopius coccus biosynthetic production of carminic acid.
Furthermore, and as known in the art, the primary glucosylated compound formed during the in vivo biosynthetic production of the glucoside end product may be an unstable intermediate compound that will not be identified in an isolated extract from D. coccus as e.g. analyzed in the above discussed article of Stathopoulou et al.
Based on the prior art, it could be speculated that a relevant primary glucosylated compound during the in vivo Dactylopius coccus biosynthetic production of carminic acid could e.g. be an unstable intermediate polyketide compound with around the same number of carbon atoms as e.g. flavokermesic acid.
According to the art—the term “glycosyltransferase” (GT) denotes a glycosyltransferase enzyme capable of transferring a sugar from an activated nucleotide sugar to an aglycon to form a glycoside.
A herein relevant DNA or amino acid sequence of a glycosyltransferase involved in the in vivo insect (Dactylopius coccus) biosynthetic pathway of carminic acid is not explicitly described in the prior art.
As known in the art, for insects that accumulate low molecular weight chemicals the relevant biosynthetic pathway genes are sometimes not present in the insect genome. For instance, some insects take up glycosides from the plants they feed on—see e.g. the article of Zagrobelny et al (Cyanogenic glucosides and plant-insect interactions; Phytochemistry. 2004 February; 65(3):293-306) or the article of Geuder et al (Journal of Chemical Ecology, Vol. 23, No. 5, 1997). Also, the relevant biosynthetic pathway genes are sometimes found in the microorganisms living in the insects, see e.g. the article of Genta et al, (Potential role for gut microbiota in cell wall digestion and glucoside detoxification in Tenebrio molitor larvae), Journal of Insect Physiology 52 (2006) 593-601.
Dactylopius coccus insects feed on cactus plants and it could be that D. coccus insects (like other insects) take up relevant glycosides from the cactus they feed on
Accordingly, based on the prior art the skilled person could not know if the genome of Dactylopius coccus actually would comprise a gene encoding a glycosyltransferase involved in the in vivo biosynthetic pathway leading to carminic acid.
WO2004/111254A1 (Poalis A/S) describes in vivo production of a glucosylated form of vanillin in e.g. eukaryotic cell yeast cells and/or prokaryotic E. coli cells by using a glucosyltransferase for conjugating glucose to the vanillin aglucon in vivo within a microorganism cell. Natural vanillin is obtained from the plant vanilla bean. Accordingly, in the prior art successful in vivo production has been described in microorganism cells of plant glycoside compounds (such as e.g. vanillin glucoside).